GSM6729704: sciATACseq_timecourse-batch1; Mus musculus; ATAC-seq
In brief, 2-5x105 cells were lysed for 3min in 500µL of RSB buffer (Tris-HCl pH 7.5 10mM, NaCl 10mM, MgCl2 3mM, NP40 0.1%, Tween-20 0.1%, digitonin 0.01%) in presence of cOmplete protease inhibitor cocktail (Roche). Next, cells were resuspended 3 times and washed in 10 mL of RSBT buffer (Tris-HCl pH 7.5 10mM, NaCl 10mM, MgCl2 3mM, Tween-20 0.1% and protease inhibitor cocktail) by inverting the tube 3 times and spinning at 500g for 10min at 4ºC. Next, the buffer was removed and 2500 nuclei were resuspended in 7.6µL of cold PBS and pipetted in a well of a 96-well plate with 1µL of i7 and i5 transposons and 10.4μL of transposition mix containing 10μL of 2xTD buffer (20 mM Tris, 10 mM MgCl2, 20% dimethylformamide, pH adjusted to 7.6), 0.2µL digitonin 1% and 0.2µL Tween 10%. The tagmentation was carried at 55ºC for 30min, nuclei were pooled and stained with 3μM 4′,6-diamidino-2-phenylindole (DAPI). The reaction was stopped by incubation at 37ºC for 15 min with 20μL 40 mM EDTA supplemented with spermidine 1mM. Next, 25 nuclei were sorted into either one or four 96 well plates (see run details) containing 11µL of EB buffer (Qiagen) and 1μL of Proteinase K 10 mg/ml (Roche). Decrosslinking was carried overnight at 65ºC and PCR was performed by adding 25µL of KAPA HiFi HotStart ReadyMix (Roche), 5μL of P5 and P7 primers (see oligonucleotides list)., 1μL of BSA 10mg/mL and 2 μL H2O. The thermocycler was set up as follows: 72ºC for 3min, 98ºC for 30s, 21 cycles of 98ºC for 10s, 63ºC for 30s,72ºC for 1min and finally 72ºC for 5min. Next, all PCR reactions were pooled and 1mL of the solution was purified using DNA clean & concentrator 5 (Zymo Research) following manufacturer instructions. Next, the library was size selected by 1x and 0.55x selection steps using AMPure XP beads (Beckman Coulter), followed by purification with QIAquick PCR purification kit (Qiagen). Libraries were sequenced with a NextSeq 550 system (Illumina) using a customized protocol 62 (genomic DNA read 1 (cycles 1–51), index 1 (transposon i7, cycles 52–59, followed by 27 dark cycles, and PCR i7, cycles 60– 67), index 2 (PCR i5, cycles 68–75, followed by 21 dark cycles, and transposon i5, cycles 76–83), and genomic DNA read 2 (cycles 84–134).